Calcareous concretions in the skin of toothed whales (Odontoceti)

Calcareous concretions in the skin of toothed whales (Odontoceti)

Günther Behrmann

from the Alfred Wegener Institute for Polar and Manne Research, Bremerhaven, FRG

Abstract

Communicated by W. Amtz Received: 5 October 1994 Accepted: 11 March 1995

The epidermis of toothed whales contains various forms of calcareous concretions, first described from some toothed whale species by Viale (1979). Concretions have now been found in the skin of other toothed whale species. Their origin was investigated using light and SE-microscopy of fresh and fixed material, and the concretions were also analysed by X-ray energy dispersion.

Kurzfassung

Kalkkonkretionen in der Haut von Zahnwalen (Odontoceti)

Die Epidermis der Zahnwale enthält verschiedene Formen von Kalkkonkretionen, die erst- mals von einigen Zahnwalarten von Viale (1979) beschrieben wurden. Jetzt wurden in frischen und fixierten Häuten anderer Zahnwalarten ebenfalls Kalkkonkretionen gefunden und deren Entstehung lichtmikroskopisch untersucht. Die separierten Kalkkonkretionen wurden auch mittels Licht- und RE-Mikroskopie untersucht und mit Hilfe der Dispersions- energie analysiert.

Resurne

Concretions calcaires dans I 'epidermes des baleines adents (Odontocetes)

L'epiderme des baleines adents (Odontocetes) contient diverses formes de concretions calcaires qui furent d'abord decrites d'apres quelques especes de baleines par Viale en 1979.

On a maintenant egalement rencontre des concretions calcaires dans des peaux fraiches et fixees d'autres especes de baleines adents et leurs formations ont ete etudiees au microscope.

Les concretions calcaires isolees ont ete ega\ement analysees a \'aide de microscopes radio- graphiques ainsi que par energie de dispersion.

Arch. Fish. Mar. Res. 43(2), 1996, 183-193, 10 figures

Introduction

The skin (integumentum commune) of cetaceans was originally regarded to be mammal- like, elastic, smooth and without any hard substances. In the ninteenth century cal- careous concretions and horny substances were discovered by Burmeister (1869) and Kükenthai (1890). Viale (1979) was the first to separate these substances and identify the single forms using electronic microscopy. The origin of the calcareous concretions and their chemical components is still not known.

The present investigation used fresh sampies, a simple maceration procedure in warm distilled water, and sophisticated technology including a scanning electron microscope and X-ray microanalyser. This resulted in clear photographs and a better identification of these substances than was hitherto possible.

Material and methods

Fresh sampies of the harbour porpoise Phocoena phocoena (L., 1758) were fixed in for- maün/glutaraldehyd (2 % I 2 %). Fresh skin segments (5 to 5 centimetres) and fixed sampies (4 per cent neutral buffered formalin) of beached toothed whales were also avail- able: One sperm whale Physeter macrocephalus L., 1758, two northern bottlenose dol- phins Hyperoodon ampullatus (Forster, 1770), three killer whales Orcinus orca (L., 1758), two bottle-nosed dolphins Tursiops truncatus (Montagu, 1821), two common dolphins Delphinus delphis L., 1758, and five harbour porpoises Phocoena phocoena (L., 1758).

The fixed material was cleaned with fresh water, impregnated with paraffin or resin, cut into histological slices 3 to 8 /Lm thick, and haematoxilin! eosin stained. The material was identified using phase contrast microscopy and different colour filters. This examination showed that the skin of all examined toothed whales contains hard substances.

After removing the blubber, fresh material was also cut and cleaned using a high pressure water beam. Thereafter the skin was macerated in warm water at 45

o e.

material was separated in distilled water by centrifugation, coated in carbon (C) and a qualitative analysis of the elements was made by energy dispersal X-ray micro-analysis (EDAX) in a scanning electron microscope (SEM). These analyses show the relation- ships of the single elements to each other. The hard substances used for SEM-photo- graphy were coated with gold.

During maceration in water all soluble substances were lost. Analyses of the following elements were used to identify individual forms of concretions: Sodium (Na), magne- sium (Mg), silicon (Si), phosphorus (P), sulphur (S), chloride (Cl), potassium (K) and calcium (Ca). Carbon (C) used for shadowing of the substances is not considered here.

Results

Calcareous corpuscles with a diameter of up to 10 /Lm originate in skeleroblasts situated in the epidermal prickle celllayer, which are identified by their big nucleus. Two forms of skeleroblasts were discernible. The first mode of calcification by calcium-phosphate

184 Arch. Fish. Mar. Res. 43(2), 1996

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Figure 1: Harbour porpoise: Calcareous spherules (the small dark points) in the parakeratotic layer of the upper lip; unstained 400x. Scale bar

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Figure 2: Common dolphin: A non identified worm (only the tail is visible) surrounded by calcareous concretions, SE photo graph. Scale bar

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Arch. Fish. Mar. Res. 43(2), 1996 185

begins to accumulate at the periphery of the cel1leading to the formation of spherules (Figs. 1 and 4). The second mode of calcification begins in the centre of the cell resulting in the formation of the central plate of a chromatophore (Fig. 9).

Small calcareous corpuscles with a diameter of nearly 1 j.Lm originate in the intercellular tissue between the prickle cells. Small and large round calcareous corpuscles combine to form larger calcareous concretions (Figs. 5 and 8) which are stored in the whole integu- ment.

Only a few concretions (Figs. 2, 17 and 18) were located in the prickle cel1layer of the epidermis, most hard concretions being located in the upper epidermallayer (parakera- totic layer; Simpson and Gardner 1972). 6000 to 8000 spherical corpuscles with

Figure 3: Harbour porpoise: Diamond-like oxalate crystals (A) are the germs of this calcareous concretion, separated in fresh water; length

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186 Arch. Fish. Mar. Res. 43(2), 1996

Calcareous concretions in the skin o[ toothed whales

Figure 4: Bottle-nosed dolphin: Foraminifera-like spherical corpuscles containing Ca, Mg, S, and Si, SE-photo graph. Scale bar

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Figure 5: Harbour porpoise: Solid calcareous concretions containing elements (Fig.10 C) nearly comparable to hard limestones, and are also very hard, SE photograph. Scale bar = 10 !Lm.

Arch. Fish. Mar. Res. 43(2), 1996 187

Figure 6: Common dolphin: Yellow pigments stored between the spiny concretions and only seen in unfixed sampies using a light microscope. SE photograph. Scale bar = 10 1J..111.

Figure 7: Harbour porpoise: Centre of the rosette-like concretion, SE photo graph. Scale bar

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188 Arch. Fish. Mar. Res. 43(2), 1996

Calcareous concretions in the skin o[ toothed whales

Figure 8: Harbour porpoise: Mushroom-like concretion, SE photograph. Scale bar = 10 f..Lm.

Figure 9: Killer whale: Calcareous disk of a melanophore, SE photograph. Scale bar

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Arch. Fish. Mar. Res. 43(2), 1996 189

diameters less than 10 /-Lm can be found per square centimetre of the parakeratotic layer (Fig.1). Using colour filters the concretions could clearly be distinguished from lipid droplets, horny substances and chromatophores.

The smallest calcareous grains, with a diameter of nearly 1 /-Lm, surrounded foreign particles, which became fully integrated into the calcareous grains forming a non- vulnerable scale (Figs. 2 and 3). Up to 24 of these calcareous concretions of various forms - with lengths of up to 300 /-Lm - can be found per square centimetre of the ventral epidermis. The elemental composition of these scales is somewhat similar to that of skull bones, although they contain less phosphorus (Fig. 10 A).

%

A

B

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20 20

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NaMgSi P S CI K C.

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NaMgSi P S CI K Ca .

Figure 10: Results of the qualitative analysis: The distribution of the elements expressed in per cent in the total mass. A: Calcareous concretions of Figs. 1,2, 3 and 8. The distribution of the elements is comparable with the beginning of an ossification in the skin of vertebrates.

B: Foraminifera-like spherical corpuscles of Fig. 4. The distribution of the elements is bone- like. C: The distribution of the elements in the solid concretion of Fig. 5 show a limes tone- like consistence. D: Prickly concretions of Figs. 6 and 7. With its high level of silicon, the spines of the spiny concretions have a hair-like structure.

190 Arch. Fish. Mar. Res. 43(2), 1996

Calcareous concretions in the skin

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Besides the spherules and scale-like concretions some other substances are located in the skin of odontocetes. Foraminifera-like spherical corpuscles (Fig. 4) with a diameter up to 15 J.Lm are frequently found in the epidermis. Some of these globular corpuscles form clusters or arrow-like forms. In these substances (Fig. 10 B) there was a high degree of calcium associated with keratotineous elements (Mg, Si, P, Sand K). Solid (Fig. 5) and foliated scales were rare. Some scales grew together forming concretions with an exten- sion of more than 0.5 mm²• There was a high portion of calcium combined with less keratotineous substances (Si, S, and P) (Fig.10 C) in both types of concretions. The functions of the solid and foliated scales are still unknown.

With a high accumulation of silicon and less calcium (Fig. 10 D), the spiny concretions (Fig.6) have a keratotineous character. On examining unfixed skin particles of the com- mon dolphin their function became clearly visible. Between the hollow spines bright yellow pigments are laid down.

The rosette-like concretions (Fig.7) are garnished with short spines. In the skin of the common dolphin yellow-red pigment granules are also found between the spines. These concretions with spines, called "micro-ursins" by Viale (1979), are found garnished with bright crystals in the white skin regions of the harbour porpoise and the killer whale.

The crystals have a shape comparable to that of uric acid. The qualitative analysis of the centrally situated substance is very similar to the analysis of the spiny concretions (Fig.10 D).

Big mushroom-shaped concretions were mostly situated in the prickle celllayer (Fig. 8).

They are conglomerates of the small calcareous concretions.

Together with the central basal plate of chromatophores (Fig. 9) and iridophores, up to 12 000 concretions per square centimetre of the epidermis of various types can be stored.

The basal plates and corpuscles of the chromatophores up to a diameter of 50 J.Lm contain a high level of calcium.

Discussion

In contrast to terrestrial mammals, the skin of the examined toothed whales possessed many very small calcareous concretions. Using colour filters they were clearly visible under the light rnicroscope, and they evidently differed from keratotineous substances.

Using scanning electron microscopy together with X-ray microanalysis the form and structure of the concretions became clearly visible. The possibility to make X -ray micro- analyses of the substances smaller than one micrometre permits the separation of cal- careous concretions from other substances also located in the skin. Together with the examinations of Viale (1979) the shapes of calcareous concretions of nine odontocete species are now known.

At nearly 270 J.Lm long (Fig.3) the compact calcareous scales which enclose foreign particles such as nematodes, sand, sponge clerites or alcionaria, are very large. Particu- larly in the harbour porpoises many of these scales were found in the ventral skin. The isolation of foreign particles is best known from bivalves during pearl formation, in contrast the skin of mammals normally discharges particles.

Arch. Fish. Mar. Res. 43(2), 1996 191

The calcareous concretions of solid (Fig. 5) and foliated concretions are strengthened by keratotineous substances. Comparable concretions were found by Viale (1979) in the skin of Cuvier's beaked whale Ziphius cavirostris Cuvier 1823. The spiny concretions (Fig.6) termed "micro-ursins" by Viale (1979), were also found in Cuvier's beaked whale. The hair-like spines and spaces between the spines of the "rosette-like" concre- tions (Fig.7) are also strengthened by keratotineous substrates. The accumulation of crystals between the spines indicates that these concretions are newly developed forms of chromatophores containing different pigments. Between the spines in spiny concre- tions in the skin of harbour porpoises and the sperm whale brown pigments are laid down, whereas the spiny concretions of the common dolphins (Fig.6) contain yellow pigments. The rosette-like concretions are white in harbour porpoises and killer whales, and brown-red in the bottle-nosed dolphins.

Foraminifera-like spherical corpuscles (Fig.4), first described in odontocetes by Viale (1979), were also found in all examined toothed whales, although their function remains unknown. So me of these concretions are located in the centre of horny scales, and it may be that the spherical concretions are remnants of a reptilian skin. However, similar spherical corpuscles are also found in invertebrates and plants (Watabe et al. 1976).

The accumulation of calcareous spherules (Fig. 1) with a diameter less than 1 J.Lm, in the parakeratotic layer indicates that they originate in the skin. The structure of larger cal- careous concretions (Fig. 8) shows that these are built up from the small spherules. The small calcareous concretions may be the germs of the reptilian-like scales which have been discovered by Burmeister (1869) and Kükenthal (1890).

Beside the calcareous concretions many kinds of metabolites were found in the skin of toothed whales, which were similar to those stored in the human skin (Harrison 1957).

But by quantitative analysis of the elements the calcareous concretions are clearly dif- ferent from uric acid crystals.

An accumulation of calcareous concretions in epidermallayers of mammals similar to

the beginning of an ossification, is not recorded. The skin skeleton of armadillos (Xenarthra) is situated in deeper layers of the integument. The existence of chromato- phores (Behrmann 1993) in the skin of mammals is also not recorded. Calcareous con- cretions and chromatophores are known from reptiles, amphibians and fishes. The skin of cetaceans contains horny substrates and hairs as well as these concretions.

The integument of cetaceans is not comparable in structure to the skin of other vertebrates; cetaceans possess an own skin structure, and contain remnants from their ancestors.

192 Arch. Fish. Mar. Res. 43(2), 1996

Calcareous concretions in the skin

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References

Behrmann, G., 1993: "Lebensraum Meer", Evolution der Wale. (3. Auflage). Alfred- Wegener-Institut für Polar- und Meeresforschung, Abt. Wissenschaftliche Sammlung/

Nordseemuseum. Bremerhaven. 99 pp.

Burmeister H., 1869: Descripci6n de cuatro especies de Delfinides de la costa argentina en el Oceano Atlantico. Buenos Aires: Anal. Mus. pub. 6. 380 pp.

Harrison, G.A., 1957: Chemical methods in clinical medicine. London: Churchill Publisher.

180 pp.

Kükenthai, W., 1890: Über die Reste eines Hautpanzers bei Zahnwalen. Anat. Anz. 5 (8):

237-240.

Simpson,].G.; Gardner, M.B., 1972: Integumentary System. In: Ridgway, S.H. (ed.): Mam- mals of the sea. SpringfieldlIll.: Ch. Thomas Publisher, p. 363-373.

Viale D., 1979: Mise en evidence d'une fonction excretrice de la peau chez certains cetaces Odontocetes et Mysticetes.]. Exp. Mar. Biol. Ecol. 40: 201-22l.

Watabe, N.; Wilbur, K.M., 1976: The mechanisms of mineralization in the invertebrates and plants. The University of South Carolina Press. 304 pp.

Acknowledgement

I am indebted to my colleague Ms Ute Bock for doing the SEM photographs and elemental analyses.

Author's address: Günther Behrmann, Alfred-Wegener-Institut für Polar-und Mee- resforschung, Columbusstr., D-27568 Bremerhaven, FRG. Fax: (+49-471) 48 31-149.

Arch. Fish. Mar. Res. 43(2), 1996 193